Method for assaying creatine kinase isozyme activity and assay reagent

ABSTRACT

A method for measuring the activity of creatine kinase MB (CK-MB) isozyme which is accurate, high in specificity, convenient, by inhibiting the activity of a mitochondria-localized creatine kinase (mCK) isozyme to avoid the influence of mCK and a measurement reagent therefor are provided.  
     A method for measuring the activity of CK-MB isozyme comprising selectively measuring the enzymatic activity of CK isozymes by an immunological inhibition method, wherein after a treatment of selectively and immunologically eliminating the enzymatic actions of CK-M subunit and mCK is performed, the activity of remaining CK is measured.

TECHNICAL FIELD

[0001] The present invention relates to a method for assaying theactivity of creatine kinase (CK) isozyme and to an assay reagenttherefor. More particularly, the present invention relates to a methodfor assaying the activity of creatine kinase MB isozyme (CK-MB) and/orthe activity of creatine kinase isozyme localized in mitochondria (mCK)and to an assay reagent therefor.

BACKGROUND ART

[0002] Human CK includes four proteins whose genes are different. Theyconsist of two kinds of proteins originated from cytoplasms [muscle-type(M-type) and brain-type (B-type) depending on where they are localized]and two kinds of proteins originated from mitochondria, sarcomeric CK(smCK) and ubiquitous CK (umCK). smCK exists in heart muscles andskeletal muscles while umCK is present in small intestines, brains, andstocmachs. The CK isozymes originated from cytoplasms consist of dimersand are classified into 3 types, CK-MM, CK-MB, and CK-BB [Y. Takagi, R.Uzawa, and K. Gomi: Rinsho Kensa (Journal of Medical Technology), Vol.32, 1309-1315 (1988)]. mCK, which exists in the form of octamer intissues, is converted with time to dimers in blood. Hereinafter, mCK ismeant to include smCK and/or umCK.

[0003] The mobilities of the isozymes in electrophoresis are arranged inthe order of mCK (octamer), and mCK (dimer)=CK-MM, CK-MB, CK-BB from thecathode. smCK and umCK have equivalent mobilities. Since mCK (dimer)shows the same mobility as that of CK-MM, mCK (dimer) in stored blood ismeasured as CK-MM by electrophoresis. Besides, there are macro-CKs,which consist of CK and an immunoglobulin connected thereto althoughthey are not classified into isozymes. They can be confirmed fromzymograms by mobility, an immune countercurrent method. The propertiesof the isozymes of CK are shown in Table 1. TABLE 1 mCK CK-1 CK-2 CK-3Dimer Octamer Subunit BB MB MM Molecular weight 82,000 82,000 82,00084,000 320,000 Tissue Brain Cardiac Skeletal muscle muscle % Inhibitionby − 50-60% + − − anti-CK-M antibody

[0004] In clinical tests, determination of total CK or CK-MB has beenwidely performed. Particularly, CK-MB is important as a marker formyocardial infarction. Determination methods of CK-MB include an EIAmethod, an immunological inhibition method, an electrophoretic method,etc.

[0005] The EIA method can measure only CK-MB with high specificitywhereas it requires a special apparatus and is not quick. Theelectrophoretic method is cumbersome to operate and requires expertness.It is necessary to obtain the ratio of CK-MB by means of a densitometerbefore results can be obtained so that this method is not quick either.The immunological inhibition method is advantageous in that quick andeasy measurement can be performed by means of an automatic analyzer.However, it has a disadvantage that it lacks specificity.

[0006] However, currently, early stage diagnosis of acute myocardiacinfarction (hereinafter, sometimes referred to as AMI for short) isdesired, so that an immunological inhibition method has been widelyused. This method uses an inhibitory antibody to human CK-M subunit todeactivate the M subunit and determines the activity of the remaining Bsubunit. Use of the method results in measurement of unintended CK-BBand mCKs (dimer and octamer) as well as CK-MB. Of these, CK-BB, whichscarcely exists in blood, may be neglected. In addition, there are notso many diseases which involve exudation of it (release of it from thetissue into blood by the destruction of tissue or the like). However,mCK is contained in the sera of healthy persons in amounts substantiallythe same as CK-MB in terms of activity [Y. Toyoda, et al.: SeibutsuButsuri Kagaku (Japanese Journal of Electrophoresis), Vol. 42, 175-179(1998) and T. Hoshino, et al., Seibutsu Butsuri kagaku (Japanese Journalof Electrophoresis), Vol. 42, Supplement No. 2, 21 (1998)]. Furthermore,the exudation of mCK occurs in the case of necrosis of cells as in liverdiseases, malignant tumors, etc., which confuses the judgment of theresults. Recently, it has been reported that the exudation of mCK occursin enteritis due to rotavirus, infant apnea, etc. [T. Hoshino, et al.;Rinsho Byori (The Japanese Journal of Clinical Pathology), 46, PlenaryAssembly Issue, 57 (1998) and F. Kanemitsu, et al.; Rinsho Byori (TheJapanese Journal of Clinical Pathology), 46, Plenary Assembly Issue, 56(1998)]. In addition, by this measurement method, about half the CK-MBactivity is also inhibited and hence the activity is calculated bymultiplying the measured value by 2. This doubles the influence of mCKactivity. That is, the method of selectively measuring the enzymaticactivity of CK isozyme by the conventional immunological inhibitionmethod measures CK-MB activity by using an anti-human CK-M inhibitoryantibody and enables convenient and quick measurement. However, thismethod also measures the activity of mCK simultaneously so that noaccurate measurement of CK-MB activity can be expected.

DISCLOSURE OF THE INVENTION

[0007] An object of the present invention is to provide a method formeasuring the activity of CK-MB which is free of the influence by mCK,accurate, high in specificity, and convenient and which permitsautomation if desired, by selectively and immunologically eliminatingthe enzymatic action of mCK. Another object of the present invention isto provide a measurement reagent for use in the measurement method.

[0008] As a result of extensive investigation, it has been found thatthe above objects can be achieved by measuring the activity of CK aftertreatment of selectively and immunologically eliminating the enzymaticactions of CK-M subunit and mCK, thus completing the present invention.

[0009] That is, in one aspect, the present invention relates to a methodfor measuring the activity of CK-MB isozyme comprising selectivelymeasuring the enzymatic activity of CK isozymes by an immunologicalinhibition method, wherein after a treatment of selectively andimmunologically eliminating the enzymatic actions of CK-M subunit andmCK is performed, the activity of remaining CK is measured.

[0010] In another aspect, the present invention relates to the methodfor measuring the activity of CK-MB isozyme as described above, whereinthe treatment of selectively and immunologically eliminating theenzymatic actions of CK-M subunit and mCK is a treatment in which aninhibitory antibody to the CK-M subunit and an inhibitory antibody tothe mCK are used.

[0011] In still another aspect, the present invention relates to themethod for measuring the activity of CK-MB isozyme as described above,wherein the inhibitory antibody to the CK-M subunit and the inhibitoryantibody to the mCK are simultaneously acted in one step.

[0012] In yet another aspect, the present invention relates to themethod for measuring the activity of CK-MB isozyme as described above,wherein the inhibitory antibody to the CK-M subunit and the inhibitoryantibody to the mCK are acted in different steps.

[0013] In a further aspect, the present invention relates to the methodfor measuring the activity of CK-MB isozyme, wherein the inhibitoryantibody to the CK-M subunit is acted and measurement of the activity ofCK is performed and then the inhibitory antibody to the mCK is acted andthe measurement is performed again to simultaneously achieve measurementof the activity of mCK.

[0014] In a still further aspect, the present invention relates to amethod for measuring the activity of mCK comprising measuring theactivity of CK, acting an inhibitory antibody to mCK, and againperforming the measurement.

[0015] In a yet further aspect, the present invention relates to ameasurement reagent for measuring CK isozymes, comprising a reagentnecessary for a measurement method as described above as a kit or as asingle item.

BRIEF DESCRIPTION OF THE ATTACHED DRAWINGS

[0016]FIG. 1 is an electrophoretograms of samples from healthy personsand mCK-positive samples treated with anti-human CK-M inhibitoryantibody and mCK inhibitory antibody.

[0017]FIG. 2 is a drawing comparing the results of the activity of CKmeasured by the control method (--; CK-MB activity+2×mCK activity) andthe activity of CK measured by the measurement method of the presentinvention (-◯-; CK-MB activity, -Δ-; mCK activity) on cases of acutemyocardiac infarction (AMI) with prognosis good.

[0018]FIG. 3 is a drawing comparing the results of the activity of CKmeasured by the control method (--; CK-MB activity+2×mCK activity) andthe activity of CK measured by the measurement method of the presentinvention (-◯-; CK-MB activity, -Δ-; mCK activity) on cases of acutemyocardiac infarction (AMI) with bad prognosis.

BEST MODE FOR CARRYING OUT THE INVENTION

[0019] The present invention has been completed based on the findingthat in a method for measuring the activity of a CK isozyme, use ofinhibitory antibodies in combination enables selective assay of theactivity of the CK isozyme.

[0020] According to a first embodiment of the present invention, themethod for assaying the activity of a CK isozyme is one for selectivelyassaying the enzymatic activity of a CK isozyme by an immunologicalinhibition method, characterized in that a sample is subjected to atreatment with an inhibitory antibody to human CK-M subunit (anti-humanCK-M inhibitory antibody) and an inhibitory antibody to human mCK(anti-human mCK inhibitory antibody) to selectively and immunologicallyeliminate the enzymatic actions of CK-M subunit and mCK and then theremaining CK activity is measured.

[0021] According to a second embodiment of the present invention, themethod for assaying the enzymatic activity of a CK isozyme is one forselectively assaying the activity of a CK isozyme by an immunologicalinhibition method, characterized in that the enzymatic actions of CK-Msubunit and mCK are selectively and immunologically eliminatedseparately and then both CK-MB activity and mCK activity are measured.That is, the method is characterized in that measurement is performed byinhibiting all the activity of CK-MM and about half the activity ofCK-MB with anti-human CK-M inhibitory antibody and then anti-human mCKinhibitory antibody is added and a further measurement is performed.

[0022] According to a third embodiment of the present invention, themethod for assaying the enzymatic activity of a CK isozyme is one forselectively assaying the activity of a CK isozyme by an immunologicalinhibition method, characterized in that the enzymatic action of mCK isselectively and immunologically eliminated and the activity of mCK ismeasured. That is, the method is characterized in that the activity ofCK is measured, an anti-human mCK inhibitory antibody is added, and thena further measurement is performed.

[0023] The basic principle of the present invention utilizes a methodfor selectively assaying the enzymatic activity of a CK isozyme by animmunological inhibition method. Generally, the assay of the enzymaticactivity of CK-MB by this method is carried out as follows. That is, anactivity inhibitory antibody specific to human CK-M subunit is used toinhibit the activity of the M subunit in MM and MB of serum CK (for MB,about half the activity is inhibited) and the remaining B subunitactivity is doubled, thereby measuring the activity of CK-MB. Themeasurement of the activity of CK-MB is carried out by an ultravioletmeasuring method utilizing an enzyme reaction system using, for example,hexokinase (HK) and glucose-6 phosphate dehydrogenase (G-6-PDH). Theassaying method is an initial rate measuring method (chemical formula 2)which measures ATP produced by a leftward reaction in the followingreaction scheme (chemical formula 1) as an increase in absorbance at 340nm of NADPH by a coupling reaction between hexokinase (HK) and G-6-PDH.

[0024] Measurement of the activity of CK-MB with an inhibitory antibodyto anti-human CK-M by a conventional method also measures the activityof mCK simultaneously so that no accurate measurement of the activity ofCK-MB can be expected. In contrast, in the present invention,measurement of the activity of CK-MB which is practically sufficientlyaccurate can be performed conveniently and quickly by inhibiting theactivity of mCK with an inhibitory antibody to anti-human MCK.

[0025] Further, the present invention can assay the activity of CK-MBconveniently and quickly by inhibiting all the activity of CK-MM andabout half the activity of CK-MB by using an anti-human CK-M inhibitoryantibody, measuring the remaining enzymatic activity of the CK isozyme,adding an anti-human mCK inhibitory antibody, and measuring the stillremaining enzymatic activity of CK isozyme. By subtracting the value ofthe CK-MB activity from the initially measured enzymatic activity of CKisozyme, the activity of mCK can be obtained.

[0026] More specifically, where the isozyme of which measurement ofactivity is intended is CK-MB, the sample is treated with an anti-humanCK-M inhibitory antibody and an anti-human mCK inhibitory antibody toinhibit the activity of the M subunit in MM and MB of serum CK and theactivity of mCK, the remaining activity of B subunit is measured andthen the measured value is doubled to obtain a CK-MB activity. Sincesubstantially no CK-BB is present in blood, this can be ignored.

[0027] On the other hand, where the CK isozyme of which measurement ofactivity is intended includes CK-MB and mCK, first the sample is treatedwith an anti-human CK-M inhibitory antibody and the activity of CK ismeasured. Then, the sample is treated with an anti-human mCK inhibitoryantibody and the activity of CK which still remains is measured againand the measured value is doubled to obtain a CK-MB activity. Bysubtracting the activity of CK obtained in the second measurement fromthe activity of CK obtained in the first measurement, the value of theactivity of mCK can be obtained. In this manner, the assaying method ofthe present invention can give both the activity of mCK and the activityof CK-MB in the same sample simultaneously.

[0028] Where only the activity of mCK is the object of measurement, theactivity of CK is measured and then the sample is treated with ananti-human mCK inhibitory antibody and the remaining activity of CK ismeasured again. By subtracting the activity of CK obtained in the secondmeasurement from the activity of CK obtained in the first measurement,the activity of mCK can be obtained.

[0029] The inhibitory antibodies used upon the activity inhibitiontreatment for removing the activity of unnecessary CK isozyme or CK-Msubunit may be used separately for treating the sample but they may beused simultaneously for treating the sample. Where the anti-human CK-Minhibitory antibody and anti-human mCK inhibitory antibody areantibodies prepared in different animal species, they may formprecipitates, etc. when they are mixed. In such a case, the antibodiesmay be used after treating one of them with affinity column in which theimmunoglobulin of the other animal is immobilized. More preferably, toavoid the risk of the existence of heterophile antibody, it isrecommended that they will be separately acted on the sample.

[0030] The inhibitory antibodies may be used for the treatment ofsamples as a reagent prepared independently of an enzyme reagent and/ora substrate solution used for the enzymatic reaction system for themeasurement of the activity of CK or as a reagent prepared by additionof them in an enzyme reagent and/or a substrate solution.

[0031] The measurement of the activity of CK can be performed by a usualassaying method using as a reagent, for example, CPK Reagent L “KOKUSAI”manufactured by International Reagents Corporation.

[0032] The anti-human CK-M inhibitory antibody and anti-human mCKinhibitory antibody used in the assaying method of the present inventioncan be obtained, for example, by immunizing goat with a human CK-Msubunit or a human mCK fraction, collecting and purifying the antibodyaccording to a conventional method [J. Schelegel et al., J. B. C., Vol.263, No. 32, pp. 16942-16953 (1988)].

[0033] Specifically, the anti-human mCK inhibitory antibody can beprepared as follows. That is, as the antigen human or mammalian mCK isused although it may vary depending on the intended specificity. Toincrease specificity, it is preferred to use species-specific antigens.

[0034] Where it is intended to obtain an antibody having affinity tohuman mCK specifically and specifically inhibiting the enzymaticactivity of human mCK, the antigen can also be prepared by geneticengineering techniques.

[0035] As the sensitizing antigen, use is made of one obtained bydissolving or suspending a purified mCK protein molecule or expressedprotein according to a partial amino acid sequence prepared by geneticengineering in a suitable buffer such as a phosphate buffer solution(PBS). The antigen liquid may be prepared to a concentration of usuallyabout 50 to about 500 μg/ml in terms of the antigen substance. In thecase of antigen substances which alone are low in antigenicity, such aspeptide antigens, it is preferred that they be connected by crosslinkingto a suitable carrier protein such as albumin or keyhole limpethemocyanin before they can be used.

[0036] The animals to be immunologically sensitized with the antigeninclude, for example, mice, rats, horses, goats, rabbits, etc.,preferably mice, and more preferably BALB/c mouse. To increase theresponse by animals to be immunized to the antigen, the above solutionmay be mixed with an adjuvant before it can be administered. Theadjuvant includes Freund's complete adjuvant (FCA), Freund's incompleteadjuvant (FIA), Ribi (MPL), Ribi (TDM), Ribi (MPL+TDM), Bordetellapertussis vaccine, muramyl dipeptide (MDP), aluminum adjuvant (ALUM),and combinations thereof. A combination of using FCA for initialimmunization and using FIA or Ribi adjuvant for additional immunizationis particularly preferably.

[0037] The immunization method maybe changed appropriately with respectto site of injection, schedule, etc. depending on the type of antigenused and whether or not it is mixed with an adjuvant. For example, wherea mouse is used as an animal to be immunized, 0.05 to 1 ml of anadjuvant-mixed antigen liquid (antigen substance: 10 to 200 μg) isadministered by intraperitoneal, subcutaneous, or intramuscularinjection, or injection in the (caudal) vein and additional immunizationis performed 1 to 4 times every about 4 to 21 days from the initialimmunization, followed by a final immunization after about 1 to 4 weeks.Where the above antigen solution is administered without using anyadjuvant, intraperitoneal injection may be used with an increased amountof antigen. The antibody dilution titer is examined by collecting bloodabout 5 to 6 days after the additional immunization. Measurement ofantibody dilution titer can be carried out by conventional methods in amanner similar to the antibody titer assay as described hereinbelow.About 3 to 5 days from the final immunization, spleen cells areseparated from the immunized animal to obtain antibody-producing cells.Where a polyclonal antibody is utilized, plasma is obtained from thecollected blood and the plasma is purified by a purification method forantibodies, known per se, with optionally adjusting the purity thereof.

[0038] The antibodies alone or in combination can specifically inhibitthe enzymatic activity of mCK in the sample to enable specificmeasurement of the activity of CK-MB. Furthermore, in the presentinvention, antiserum per se or purified IgG antibody, further Fabfragment obtained by digesting an antibody with papain can be used.

[0039] Samples measured by the present invention are not particularlylimited and those used in the art in methods for measuring CK-MB may beused.

[0040] In the present invention, a reagent for assaying the activity ofCK isozyme constituted by reagents necessary for the measurement methodof the present invention as a kit or as a single item. As the reagentreferred to herein, a reagent for assaying CK-MB which is used for acutemyocardiac infarction may be used as a part thereof. However, thepresent invention is not limited thereto.

EXAMPLES

[0041] The following examples will explain the present inventionconcretely. However, the present invention is not limited thereto.

Example 1 Preparation Method for Polyclonal Antibody

[0042] (1) Rabbit

[0043] NZW/clean (Carey) rabbits, male, weighing 1.25 to 1.35 kg/animalwhen they were procured, were raised in an animal raising chamber onstandard pellets with free water supply.

[0044] (2) Preparation of Immunogen

[0045] Human mCK was purified from a human cardiac muscle tissue and ahuman gastric tissue according to the methods described in R. Roberts etal., J. B. C., Vol. 255, pp. 2870-2877 (1980) and A. M. Grace et al., J.B. C., Vol. 258, pp. 15346-15354 (1983). From about 300 g of a humancardiac muscle was obtained purified smCK. Similarly about 150 g of ahuman stomach was obtained purified umCK. These were cryopreserved untilthey were used.

[0046] (3) Immunization Method

[0047] Antigens smCK and umCK prepared in (2) above were each adjustedto 100 μg/ml with RiBi Adjuvant (MPL+TDM) and vigorously mixed toprepare homogeneous suspensions. Then, the suspensions were eachadministered to three rabbits at the groins each in 200 μl and at twosites on the back each in 50 μl. Moreover, the above antigen adjusted inthe same manner as above was repeated administered every four weeks intotal four times.

[0048] (4) Measurement of Antibody Titer

[0049] Upon measurement of antibody titers, a small amount of wholeblood was periodically collected from the auricular veins of rabbits andsera were separated therefrom. Then, the sera were treated at 55° C. for60 minutes to inactivate the activity of CK in the leporine sera andcryopreserved until use. From the onset of immunization, antibody titersagainst smcK and umCK were examined by an mCK enzymatic activityinhibitory antibody method.

[0050] More specifically, the serum from each rabbit was diluted100-folds with PBS to prepare 50 μl of an antibody solution. This wascollected in wells of a 96-well microtiter plate, to which was added 50μl of an mCK enzyme solution (PBS buffer containing 0.2 U/ml of smCK orumCK), and the mixture was left to stand at room temperature for 10minutes. Thereafter, 100 μl of a CK coloring reagent [100 mM imidazole,2 mM EDTA, 10 mM magnesium acetate, 2 mM adenosine-5′-diphosphate (ADP),5 mM adenosine-5′-monophosphate (AMP), 40 μMP1,P5-di(adenosine-5′)-pentaphosphate (AP5A), 30 mM 1-thioglycerol, 20mM D-glucose, 2 mM NADP, 3 U/ml hexokinase, 2 U/ml glucose-6-phosphatedehydrogenase, 30 mM creatine phosphate, 1 mg/ml Nitro Blue Tetrazolium,3 U/ml diaphorase, pH 6.6] was added and the mixture was allowed toreact at 37° C. for 10 minutes.

[0051] Subsequently, each of the above wells was added 50 μl of 0.2 Nhydrochloric acid to stop the reaction and absorbance at 570 nm wasmeasured using pure water as a control. Furthermore, where the obtainedabsorbance suggested inhibition of the enzymatic activity of mCK, thechange in absorbance is small due to suppressed substrate reaction andthe existence of an enzymatic activity inhibiting specific antibodycould be identified. To note, as a negative control of antibody, the onewhich contains only PBS was used and as a blind control for sample, PBSwas used instead of the mCK enzyme solution.

[0052] (5) Study of Reaction Specificity

[0053] Enzyme solutions containing optimal concentrations of human CK-MBand human CK-MM, respectively, were prepared and the anti-human smCKinhibitory antibody and umCK inhibitory antibody obtained as describedabove were examined for their inhibition of the enzymatic activityagainst the respective enzymes in the same manner as described above.

Example 2

[0054] The following reagents were prepared as CK activity assayingreagents.

[0055] Enzyme reagent: 140 mM imidazole, 2.8 mM EDTA, 14 mM magnesiumacetate, 2.8 mM adenosine-5′-diphosphate (ADP), 7 mMadenosine-5′-monophosphate (AMP),

[0056] 14 μM P1,P5-di(adenosine-5′) pentaphosphate (AP5A), 42 mM1-thioglycerol, 28 mM D-glucose, 2 mM NADP, 4.2 U/ml hexokinase, 2.1U/ml glucose-6-phosphate dehydrogenase, pH 6.6

[0057] Substrate solution: 150 mM disodium creatine phosphate

[0058] Fractionation reagent: 1 U/ml anti-human smCK inhibitory antibody(rabbit) or umCK inhibitory antibody (rabbit) was added to the enzymereagent to prepare a fractionation liquid.

[0059] To 20 μl each of 5-level dilutions of purified human CK(physiological saline containing 0.1% BSA) was added 250 μl of theenzyme reagent and the mixture was kept at a constant temperature of 37°C. Thereafter, it was measured of a change in absorbance at a wavelengthof 340 nm (A). Furthermore, 2 to 3 minutes after the addition of 100 μlof the substrate solution, a change in absorbance was also measured (B).The activity of CK was calculated according to the following equation(mathematical formula 1).

[0060] Mathematical Formula 1${{CK}\quad {Activity}\quad \left( {U/L} \right)} = \frac{\left( {{370 \times B} - {270 \times A}} \right) \times 1000}{6.3 \times 20}$

[0061] Next, similar operation was made using the fractionation reagentinstead of the enzyme reagent to measure a change in absorbance and thusthe activity of CK. The activity of CK was calculated by the aboveequation (mathematical formula 1). The results obtained are shown inTables 2 to 4. The antibody inhibited human mCK but not inhibited CK-Msubunit and CK-B subunit. TABLE 2 Influence of an inhibitory antibody onthe activity of CK-MM (U/L). Dilution 0 1/5 2/5 3/5 4/5 5/5 Anti-mCKAntibody absent 0 752 1496 2262 3010 3748 Anti-smCK Antibody present 0747 1496 2250 2998 3740 Anti-umCK antibody present 0 750 1490 2258 30023745

[0062] TABLE 3 Influence of an inhibitory antibody on the activity ofCK-BB (U/L). Dilution 0 1/5 2/5 3/5 4/5 5/5 Anti-mCK Antibody absent 128 57 85 115 143 Anti-smCK Antibody present 0 30 58 87 114 140 Anti-umCKAntibody present 0 30 61 87 116 143

[0063] TABLE 4 Influence of an inhibitory antibody on the activity ofCK-MM (U/L) and the activity of umCK (U/L) Dilution 0 1/5 2/5 3/5 4/55/5 smCK Anti-mCK Antibody absent 1 31 64 95 124 157 Activity Anti-smCKAntibody present 0 1 0 1 1 1 Anti-umCK antibody present 0 2 3 3 4 4 UmCKAnti-mCK Antibody absent 0 25 51 75 101 128 Activity Anti-smCK Antibodypresent 0 1 1 2 4 3 Anti-umCK antibody present 1 0 1 2 1 2

[0064] Inhibition cross-reactivity was observed regardless of whetherthe antibody was prepared using smCK or umCK. Therefore, the followingexamples were practiced using an anti-human smCK inhibitory antibody. Inthe following examples, the antibody expressed anti-human mCK inhibitoryantibody is anti-human smCK inhibitory antibody having the inhibitioncross-reactivity.

Example 3

[0065] To 100 μl each of a sample from a healthy human and humanmCK-positive sample was added 10 μl each of physiological saline,anti-human CK-M inhibitory antibody (goat), anti-human mCK inhibitoryantibody (rabbit), or a mixture of the two inhibitory antibodies andelectrophoresis was performed for 40 minutes using Pol-E-Film System™(Agarose electrophoresis). After the electrophoresis, the CK coloringreagent prepared in Example 1 was infiltrated into the electrophoresesgel, which was incubated at 37° C. for 30 minutes. After stopping thereaction with an aqueous 5% acetic acid solution and washing withpurified water, the gel was dried and copied. The results are shown inFIG. 1. The results suggest the anti-human CK-M inhibitory antibodyalone does not inhibit mCK so that mCK will be measured as CK-MB andthat use of the anti-human CK-M inhibitory antibody and anti-human mCKinhibitory antibody in combination enables specific measurement ofCK-MB.

Example 4

[0066] A formulation (control method) was prepared by adding 1.0 U/ml ofan anti-human CK-M inhibitory antibody (goat) to the enzyme reagent ofExample 2 and another formulation (the present invention) was preparedby adding 1.0 U/ml of an anti-human CK-M inhibitory antibody (goat) andfurther 1 U/ml of an anti-human mCK inhibitory antibody (rabbit) to theenzyme reagent of Example 2. Nineteen (19) samples showing a CK activityof 300 U/L or less and 26 samples from patients suffering liver disease,showing a GPT activity of 80 U/L or more were measured of the activityof CK-MB by the operation method of Example 2 and comparison was made.The activity of CK-MB was calculated by the following equation(mathematical formula 2).

[0067] Mathematical Formula 2${{CK} - {{MB}\quad {Activity}\quad \left( {U/L} \right)}} = \frac{\left( {{370 \times B} - {270 \times A}} \right) \times 1000 \times 2^{*}}{6.3 \times 20}$

[0068] 2*: Factor converting CK-B activity into CK-MB activity.

[0069] The results are shown in Tables 5 and 6. From the results shownin Table 5, it revealed that the average value of CK-MB activity was 13U/L in the control method in contrast to 7 U/L in the present invention,which was about the half of the former. Conventionally, the cut-offvalue of CK-MB activity has been said to be about 25 U/L. However, inthe present invention, the activity of mCK which reacts non-specificallycan be inhibited so that the cut-off value can be set to about 10 U/L.

[0070] As shown in Table 6, among the samples from patients sufferingliver disease, 7 samples showed an activity of 25 U/L or more in thecontrol method though they were not samples from patients sufferingacute myocardiac infarction. However, in the present invention, all thesamples showed an activity of 10 U/L or less.

[0071] From the above results, it is expected that due to a decrease inthe cut-off value of CK-MB activity, the present invention will providea higher sensitivity and specificity as an early marker for acutemyocardiac infarction than conventional measurment method.

Example 5

[0072] Anti-human mCK inhibitory antibody may be added to the enzymereagent or to the substrate solution. Furthermore, it is possible toprepare an anti-human mCK inhibitory antibody solution as a separatereagent from the substrate solution and the enzyme reagent, performconventional measurement of the activity of CK-MB using the enzymereagent containing an anti-CK-M inhibitory antibody and the enzyme, addthe anti-human mCK inhibitory antibody solution, and then measure theactivity of mCK, followed by subtracting the activity of mCK from theactivity of CK-MB. The advantage in this case is that the activity ofmCK besides the activity of CK-MB can be obtained. Hereinafter, anexample thereof will be described.

[0073] Blood was periodically collected from patients whose prognosiswas good and those whose prognosis was bad and were dead after the onsetof acute myocardiac infarction. The samples were used for simultaneousmeasurement of the activity of CK-MB and the activity of mCK.

[0074] To 20 μl of each sample was added 250 μl of a reagent obtained byadding the anti-human CK-M inhibitory antibody to the enzyme reagent ofExample 2 and the mixture was kept at a constant temperature of 37° C.Thereafter, the mixture was measured of a change in absorbance at awavelength of 340 nm (A). Next, 100 μl of the substrate solution wasadded and after 2 to 3 minutes, a change in absorbance was measured (B).Then, 50 μl of the anti-human mCK inhibitory antibody (100 mM imidazolebuffer containing 5.7 U/ml, pH 6.6) and after 2 to 3 minutes, a changein absorbance was measured (C). The activity of CK-MB and the activityof mCK were calculated by the following equation (mathematical formula3). They were compared with the activity of CK-MB obtained by thecontrol method. The results are shown in Tables 7and 8 and FIGS. 2 and3.

[0075] From the results, it can be seen that in cases where theprognosis was good, no difference was observed between the controlmethod and the assay method of the present invention until the peak wasreached after the onset of acute myocardiac infarction while in therecovery phase where the activity of CK-MB decreased, the assay methodof the present invention gives lower values, indicating faster recovery.

[0076] It is believed that this is because in an early phase, the CK-MBoriginated from cytoplasm exudes and thereafter exudation of the mCKoriginated from mitochondria occurs along with the necrosis of cells.Thus, it is understood that usefulness of the method of the presentinvention in observing the course of prognosis is increased. In caseswhere the prognosis was good, the activity of mCK changes to a lowerlevel while in cases where the prognosis is bad, the activity of mCKchanges to a higher level.

[0077] Since enzymes in mitochondria will not exude in blood until thenecrosis of cells occurs, currently mGOT (mitochondria glutamicacid-oxaloacetic acid transaminase) activity is measured. However, useof the present invention makes it possible to accurately measure theactivity of CK-MB and at the same time obtain information on theseverity of acute myocardiac infarction and on the prognosis by analogywith the degree of necrosis of cells.

[0078] Mathematical Formula 3${{CK} - {{MB}\quad {Activity}\quad \left( {U/} \right)}} = \frac{\left( {{420 \times C} - {270 \times A}} \right) \times \text{1,000} \times 2^{*}}{6.3 \times 20}$

[0079] 2*: Factor converting CK-B activity into CK-MB activity.${{mCK}\quad {Activity}\quad \left( {U/L} \right)} = \frac{\left( {{370 \times B} - {420 \times C}} \right) \times \text{1,000}}{6.3 \times 20}$

TABLE 5 Results of measurement of CK-MB activity of samples having a CKactivity of 300 U/L or less Sample No. Control method Invention 1 8.93.7  2 10.9 5.1  3 10.0 5.0  4 16.9 8.7  5 8.4 2.9  6 22.5 10.0  7 13.87.3  8 17.0 8.5  9 10.8 5.8 10 18.3 9.0 11 9.9 5.4 12 6.7 3.2 13 19.19.9 14 9.6 4.8 15 19.4 8.9 16 15.2 9.0 17 8.6 3.4 18 9.0 6.0 19 17.2 9.1Average 13.3 6.7 SD 4.7 2.4 Average + 2SD 22.7 11.5 Average − 2SD 3.91.9

[0080] TABLE 6 Results of measurement of CK-MB activity of sampleshaving a GPT activity of 80 U/L or more Sample No. Control methodInvention  1 11.4 6.9  2 5.1 2.0  3 8.0 4.8  4 5.8 2.6  5 6.8 3.2  618.2 7.7  7 11.4 5.3  8 35.7 9.0  9 4.4 2.0 10 19.7 8.8 11 10.4 4.9 1213.3 5.2 13 27.9 9.3 14 11.5 6.7 15 8.5 4.0 16 25.1 8.8 17 10.2 4.9 186.9 3.0 19 10.5 4.5 20 42.8 7.5 21 8.7 4.5 22 30.6 5.7 23 34.6 8.9 246.7 2.6 25 22.8 8.2 26 47.9 9.4 Average 17.1 5.8 SD 12.5 2.5 Average +2SD 42.1 10.8 Average − 2SD −7.9 0.8

[0081] TABLE 7 Cases with good prognosis Time after Control methodInvention Onset (CK − MB) + 2 × mCK CK − MB mCK 4 42 31 4 6 90 80 4 8190 175 6 10 325 310 5 12 486 475 6 14 480 460 8 18 380 338 18 21 313276 20 24 235 191 22 33 165 105 28 45 62 12 25 63 40 11 14 75 23 10 7 9915 5 5

[0082] TABLE 8 Cases with bad prognosis Time after Control methodInvention Onset (CK − MB) + 2 × mCK CK − MB mCK 2 32 22 4 4 64 51 4 7201 191 4 9 385 371 6 13 532 515 10 16 610 570 18 20 630 536 45 24 598385 104 28 578 266 154 34 460 145 155 42 390 70 158

[0083] Industrial Applicability

[0084] Currently, the specificity of measurement of the activity ofCK-MB is increased by addition of an antibody that specifically inhibitsthe activity of CK-MM to the reagent system. However, the activity ofmCK is also measured so that the activity of CK-MB specific to the oneoriginated from cardiac muscle is not always measured. In the presentinvention, by using an anti-human mCK inhibitory antibody tospecifically inhibit the activity of mCK which has not beendistinguished conventionally, it is possible to make an accuratemeasurement of the activity of CK-MB. The assay method of the presentinvention uses an anti-human mCK inhibitory antibody and specificallyinhibit the activity of mCK in the serum of patients suffering acutemyocardiac infarction, thereby measuring true CK-MB activity. This makesit possible to grasp severity of acute myocardiac infarction andpathological state more credibly. The present invention enables not onlyearly diagnosis but also monitoring of its therapy of acute myocardiacinfarction so that it has a great importance in clinical tests.

1. A method for measuring the activity of creatine kinase (CK) MB(CK-MB) isozyme comprising selectively measuring the enzymatic activityof CK isozymes by an immunological inhibition method, wherein after atreatment of selectively and immunologically eliminating the enzymaticactions of CK-M subunit and mCK is performed, the activity of remainingCK is measured.
 2. The method for measuring the activity of CK-MBisozyme as claimed in claim 1, wherein the treatment of selectively andimmunologically eliminating the enzymatic actions of CK-M subunit andmCK is a treatment in which an inhibitory antibody to the CK-M subunitand an inhibitory antibody to the mCK are used.
 3. The method formeasuring the activity of CK-MB isozyme as claimed in claim 2, whereinthe inhibitory antibody to the CK-M subunit and the inhibitory antibodyto the mCK are simultaneously acted in one step.
 4. The method formeasuring the activity of CK-MB isozyme as claimed in claim 2, whereinthe inhibitory antibody to the CK-M subunit and the inhibitory antibodyto the mCK are acted in different steps.
 5. The method for measuring theactivity of CK-MB isozyme as claimed in claim 2, wherein the inhibitoryantibody to the CK-M subunit is acted and inhibitory antibody to the mCKis acted and the measurement is performed again to simultaneouslyachieve measurement of the activity of mCK.
 6. A method for measuringthe activity of mCK comprising measuring the activity of CK, acting aninhibitory antibody to mCK, and again performing the measurement.
 7. Ameasurement reagent for measuring CK isozymes, comprising a reagentnecessary for a measurement method as claimed in any one of claims 1 to6 as a kit or as a single item.